The long term goal of this research is to determine in detail how various O2 oxidoreductases catalyze reactions of O2. Since these enzymes are necessary for so many important metabolic pathways (including: inositol catabolism, aromatic amino acid metabolism, catecholamine biosynthesis and degradation, steroid hormone biosynthesis, drug metabolism, etc.), it is felt that a more detailed understanding of their mechanisms could lead to the control or alleviation of many metabolic defects. The three enzymes listed below will be investigated during the coming grant period, but the major emphasis will be on the first. myo-Inositol oxygenase from pig kidney. This enzyme is responsible for the first committed step in inositol catabolism in animals and is known to be deficient in animals with diabetes. Since inositol derivatives are now known to act as second messengers for various hormones, a detailed study of the enzyme that is partially responsible for the inositol status of cells seems warranted. A major focus of the proposed work will be to identify and characterize enzymic groups involved in catalysis, and hopefully obtain detailed structural information about the enzyme. To this end we plan to: further characterize and develop affinity labels for the enzyme's active site residues, obtain the complete amino acid sequence of the enzyme by cloning and sequencing the enzyme's cDNA, characterize the metal ion and its binding site, hopefully obtain crystals suitable for a complete X-ray structure determination, etc. 4-Hydroxyphenylpyruvate dioxygenase from pig liver. This enzyme, which participates in the metabolism of tyrosine in all animals, is involved in several metabolic diseases. Most of the proposed experiments are aimed at identifying, either possible intermediates in the reaction, or amino acid residues at the active site. D-Aspartate oxidase from beef kidney. This peroxisomal enzyme is believed to function in the control of metabolism in animals. Our goal in the proposed research is to determine by kinetic methods (especially employing stopped flow techniques) the explanation for an unusual activation of this monomeric flavoenzyme by some effectors.